Project Summary: GABA-A receptors mediate the majority of rapid inhibitory synaptic transmission in the vertebrate central nervous system. They play a key role in cognition and are also a central figure in epilepsy and related seizure disorders. Clearly, if we wish to either develop a deep understanding of these phenomena, or design more effective drugs that target the GABA-A receptor, it is requisite that we first decipher the most basic process underlying all of these processes, the interaction between GABA-A receptors and their ligands. Therefore, the long-term goal of the P.l.'s laboratory is to develop a biophysical description of the interaction between the GABA-A receptor and its endogenous ligand, gamma-aminobutyric acid (GABA). This study proposes to employ an array of techniques (site-directed mutagenesis, patch-clamp recording, rapid ligand-application, single channel recording, kinetic modeling, and double-mutant cycle analysis) to measure the actual binding and unbinding rates for ligands in both wild type and mutant GABA-A receptors. This is a powerful new approach that should reveal detailed information regarding the structure of the ligand-binding pocket and the position and orientation of ligand molecules bound to the receptor. These results will supply detailed information needed to improve molecular and dynamic models that can be used in intelligent drug design. In addition, this study will also demonstrate the power of combining mutagenesis with kinetic analysis, hopefully increasing the number of investigators that take advantage of this synergy. Relevance GABA-A receptors are ligand-gated ion channels that are thought to be present on every neuron in the mammalian brain. They play a key role in epilepsy and are the target of myriad clinically relevant drugs including anesthetics, anti-convulsants, hypnotics (sleep aids), and anxiolytics (anti-anxiety drugs). The experiments proposed here will help us to better understand how the neurotransmittter that activates GABA- A receptors interacts with its binding site and should provide a significant step towards designing safer and more effective drugs. [unreadable] [unreadable] [unreadable]